For optimal activation and clonal expansion T-cells require both an antigen-specific signal mediated via their T-cell receptor (TCR) and a second signal, provided by accessory molecules and mediated by co-stimulatory pathways. In the absence of costimulation, TCR crosslinking by antigen does not lead to successful activation but instead leads to a state of antigen-specific unresponsiveness, termed anergy in vitro and tolerance in vivo. Mounting evidence demonstrates the biologic significance of costimulation in the antigen-specific response and underscores its role in the generation of tumor immunity, allograft rejection, and autoimmunity. However, the biochemical signals that accompany the generation of anergy and its prevention are not presently understood. Anergic cells are incapable of activating IL-2 transcription when restimulated with antigen even in the presence of costimulation. This event is associated with lack of activation of lck, ZAP-70, Ras, ERK, JNK, and defective transactivation of the IL-2 enhancer elements AP-1 and NF-AT. Therefore, the question arises whether the anergic state results from the absence of sufficient positive signals or, alternatively, from the activation of distinct signaling pathways. Recently, this investigator has identified a novel signaling pathway that results in active blockade of IL-2 gene transcription in T-cell anergy. Anergic cells demonstrate increased phosphorylation of fyn-co-immunoprecipitated cbl, recruitment of crkL/C3G complexes and activation of Rap 1, a competitor of Ras. Transfection of even low levels of the active form of Rap 1 in Jurkat T-cells recapitulates the anergic defect and results in blockade of TCR and CD28 mediated IL-2 gene transcription. These results strongly argue that a distinct signaling pathway is induced by TCR signaling in the absence of adequate costimulation which induces T-cell anergy and active inhibition of IL-2 gene transcription. Understanding of the precise downstream end points of the anergic mediators will provide insights in the potential signaling defects of helper T-cells in tumor bearing hosts which need to be reversed in order to generate tumor-specific immune response. Moreover, such knowledge will facilitate the ability to identify the specific molecular targets of anergy and design novel clinical therapeutic approaches in order to induce antigen-specific transplantation tolerance. To achieve these goals two aims are proposed: First, to identify known and yet to be described molecules that are associated with activated Rap 1 and study their role in the induction of T-cell anergy; second, to study the mechanism(s) of how Rap 1-GTP and its associated molecules inhibit transcriptional activation of the IL-2 gene.